A fundamental question in addiction biology is why opiate alkaloid drugs such as morphine and heroin have a high liability for inducing tolerance and addiction while endorphins and enkephalins, the native peptide ligands for opioid receptors do not. Following activation by agonists, opioid receptors are regulated by multiple mechanisms. Of these regulatory mechanisms, rapid endocytosis of opioid receptors is of particular interest because it is differentially regulated by peptide agonists and alkaloid drugs. Specifically, endogenous opioid peptides and certain opiate drugs such as etorphine and methadone stimulate the rapid internalization of mu opioid receptors. Morphine however, strongly activates receptor signaling but fails to stimulate the rapid internalization of mu opioid receptors. Furthermore, following endocytosis, individual receptors can be sorted differentially between recycling endosomes and lysosomes. This sorting mechanism can contribute to receptor regulation in two ways that have opposing effects on cell signaling. First, endocytosis can serve as a mechanism for receptor resensitization by delivering internalized receptors to endosomes from where they are recycled to the plasma membrane in a fully active state. Second, rapid internalization can serve as a first step toward receptor downregulation by delivering the receptors to endosomes from which they are sent to lysosomes for degradation. Most membrane proteins are rapidly recycled, presumably by default, because membrane itself is recycled continuously. Therefore it is likely that membrane receptors that are rapidly degraded following their endocytosis do so through a specific targeting mechanism. The post-endocytic sorting of individual receptors between recycling and degradative fates is biochemically specific and appears to be highly regulated, identifying this sorting step as a fundamental mechanism that controls the degradative down-regulation of a large number of receptors relevant to neuropsychiatric research. Hence for each receptor/ligand pair, one must evaluate both the endocytic and post-endocytic properties. The specific aims outlined below are designed to elucidate the molecular basis for endocytosis and sorting of the opioid receptors and assess in a cell culture model what effects altered trafficking has on the development of tolerance and withdrawal.